Principles of Fluorescence Spectroscopy

608 MULTIPHOTON EXCITATION AND MICROSCOPY
Figure 18.1. Jablonski diagram for one-, two-, and three-photon excitation.
Figure 18.2. Schematic comparison of one- and two-photon excitation.
multiphoton absorption. Hence we can still use a Jablonski
diagram with S 1 emission to describe multiphoton excitation.
The quadratic or higher-order dependence of MPE on
the incident intensity is a favorable property for optical
imaging. Assume a wavelength for 1PE is incident on a
Figure 18.3. Comparison of one-photon excitation (blue arrow) and
two-photon excitation (red arrow) of a fluorescein solution. Courtesy
of Dr. Peter T. C. So from the Massachusetts Institute of Technology.
cuvette (Figure 18.2). The amount of light absorbed in any
plane at a distance is proportional to the incident intensity
at this plane. Focusing a beam on the center of a cuvette
changes the size of the beam but does not change the total
amount of light passing through a plane at a position x. The
emission intensity is constant at all positions x across the
cuvette, assuming the absence of inner-filter effects (top).
Now consider 2PE with a longer wavelength. The
amount of light absorbed is proportional to the square of the
intensity. Focusing the beam decreases its size but increases
its intensity. As a result the amount of light absorbed is
not constant across the cuvette, but shows a maximum at the
focal point where the incident intensity is highest (Figure
18.2, bottom). This effect can result in strongly localized
excitation. Figure 18.3 shows a fluorescein solution illuminated
with wavelengths for 1PE and 2PE. For 1PE the fluorescein
is excited across the cuvette. For 2PE the fluorescein
is only excited in a small spot at the focal point of the
laser beam.
Most fluorophores photobleach rapidly in fluorescence
microscopy. Localized excitation is an advantage under
these conditions. If a biological sample undergoes 1PE the
light is absorbed at all depths in the sample, not just in the
focal plane. 11–12 As a result the entire thickness of the sample
undergoes photobleaching (Figure 18.4, left) and photodamage
occurs across the entire thickness of the sample.
Figure 18.4. Photobleaching of rhodamine in a Formvar layer with one- (left) and two-photon excitation (right). From [12].